Rapid isolation of pure Complement Factor H from serum for functional studies by the use of a monoclonal antibody that discriminates FH from all the other isoforms

Several mutations have been identified in the gene coding for Complement Factor H (FH) from patients with atypical Hemolytic Uraemic Syndrome (aHUS), Age-related Macular Degeneration (AMD) and Membranoproliferative Glomerulonephritis (MPGN). These data allow for a precise description of the structural changes affecting FH, but a simple test for specifically assessing FH function routinely is not yet of common use. We have produced and characterised a monoclonal antibody (5H5) which discriminates between FH and the smaller FH-like 1 and FH-related proteins and show here that it specifically binds to FH without detecting the smaller isoforms. We therefore used this mAb for a quick, one-step micro-purification of FH directly from control sera and showed that this affinity chromatography procedure is not disruptive of its cofactor function. We also developed a modified sheep erythrocytes haemolysis test using our antibody and affinity-purified FH. These tests can be used in conjunction for assessing the function of FH purified from patients affected by FH-related diseases. Moreover we used this mAb to develop a FH-specific ELISA test

Characterization of a new trabectedin-resistant myxoid liposarcoma cell line that shows collateral sensitivity to methylating agents

Myxoid Liposarcomas (MLS), characterized by the expression of FUS-CHOP fusion gene are clinically very sensitive to the DNA binding antitumor agent, trabectedin. However, resistance eventually occurs, preventing disease eradication. To investigate the mechanisms of resistance, a trabectedin resistant cell line, 402-91/ET, was developed. The resistance to trabectedin was not related to the expression of MDR related proteins, uptake/efflux of trabectedin or GSH levels that were similar in parental and resistant cells. The 402-91/ET cells were hypersensitive to UV light because of a nucleotide excision repair defect: XPG complementation decreased sensitivity to UV rays, but only partially to trabectedin. 402-91/ET cells showed collateral sensitivity to temozolomide due to the lack of O(6) -methylguanine-DNA-methyltransferase (MGMT) activity, related to the hypermethylation of MGMT promoter. In 402-91 cells chromatin immunoprecipitation (ChIP) assays showed that FUS-CHOP was bound to the PTX3 and FN1 gene promoters, as previously described, and trabectedin caused FUS-CHOP detachment from DNA. Here we report that, in contrast, in 402-91/ET cells, FUS-CHOP was not bound to these promoters. Differences in the modulation of transcription of genes involved in different pathways including signal transduction, apoptosis and stress response between the two cell lines were found. Trabectedin activates the transcription of genes involved in the adipogenic-program such as c/EBPα and β, in 402-91 but not in 402-91/ET cell lines. The collateral sensitivity of 402-91/ET to temozolomide provides the rationale to investigate the potential use of methylating agents in MLS patients resistant to trabectedin

A systems biology approach to investigate the mechanism of action of trabectedin in a model of myelomonocytic leukemia

This study was designed to investigate the mode of action of trabectedin in myelomonocytic leukemia cells by applying systems biology approaches to mine gene expression profiling data and pharmacological assessment of the cellular effects. Significant enrichment was found in regulons of target genes inferred for specific transcription factors, among which MAFB was the most upregulated after treatment and was central in the transcriptional network likely to be relevant for the specific therapeutic effects of trabectedin against myelomonocytic cells. Using the Connectivity Map, similarity among transcriptional signatures elicited by treatment with different compounds was investigated, showing a high degree of similarity between transcriptional signatures of trabectedin and those of the topoisomerase I inhibitor, irinotecan, and an anti-dopaminergic antagonist, thioridazine. The study highlights the potential importance of systems biology approaches to generate new hypotheses that are experimentally testable to define the specificity of the mechanism of action of drugs.The Pharmacogenomics Journal advance online publication, 13 December 2016; doi:10.1038/tpj.2016.76

Pattern and quality of care of cancer pain management. Results from the Cancer Pain Outcome Research Study Group

Most patients with advanced or metastatic cancer experience pain and despite several guidelines, undertreatment is well documented. A multicenter, open-label, prospective, non-randomised study was launched in Italy in 2006 to evaluate the epidemiology, patterns and quality of pain care of cancer patients. To assess the adequacy of analgesic care, we used a standardised measure, the pain management index (PMI), that compares the most potent analgesic prescribed for a patient with the reported level of the worst pain of that patient together with a selected list of clinical indicators. A total of 110 centres recruited 1801 valid cases. 61% of cases were received a WHO-level III opioid; 25.3% were classified as potentially undertreated, with wide variation (9.8–55.3%) according to the variables describing patients, centres and pattern of care. After adjustment with a multivariable logistic regression model, type of recruiting centre, receiving adjuvant therapy or not and type of patient recruited (new or already on follow-up) had a significant association with undertreatment. Non-compliance with the predefined set of clinical indicators was generally high, ranging from 41 to 76%. Despite intrinsic limitations of the PMI that may be considered as an indicator of the poor quality of cancer pain care, results suggest that the recourse to WHO third-level drugs still seems delayed in a substantial percentage of patients. This delay is probably related to several factors affecting practice in participating centres and suggests that the quality of cancer pain management in Italy deserves specific attention and interventions aimed at improving patients' outcomes

Evaluation of volumetric modulated arc therapy (VMAT) with Oncentra MasterPlan® for the treatment of head and neck cancer

Background Several comparison studies have shown the capability of VMAT to achieve similar or better plan quality as IMRT, while reducing the treatment time. The experience of VMAT in a multi vendor environment is limited. We compared the plan quality and performance of VMAT to IMRT and we investigate the effects of varying various user-selectable parameters. Methods IMRT, single arc VMAT and dual arc VMAT were compared for four different head-and-neck tumors. For VMAT, the effect of varying gantry angle spacing and treatment time on the plan quality was investigated. A comparison of monitor units and treatment time was performed. Results IMRT and dual arc VMAT achieved a similar plan quality, while single arc could not provide an acceptable plan quality. Increasing the number of control points does not improve the plan quality. Dual arc VMAT delivery time is about 30% of IMRT delivery time. Conclusions Dual arc VMAT is a fast and accurate technique for the treatment of head and neck cancer. It applies similar number of MUs as IMRT, but the treatment time is strongly reduced, maintaining similar or better dose conformity to the PTV and OAR sparing

Rotational IMRT techniques compared to fixed gantry IMRT and Tomotherapy: multi-institutional planning study for head-and-neck cases

<p>Abstract</p> <p>Background</p> <p>Recent developments enable to deliver rotational IMRT with standard C-arm gantry based linear accelerators. This upcoming treatment technique was benchmarked in a multi-center treatment planning study against static gantry IMRT and rotational IMRT based on a ring gantry for a complex parotid gland sparing head-and-neck technique.</p> <p>Methods</p> <p>Treatment plans were created for 10 patients with head-and-neck tumours (oropharynx, hypopharynx, larynx) using the following treatment planning systems (TPS) for rotational IMRT: Monaco (ELEKTA VMAT solution), Eclipse (Varian RapidArc solution) and HiArt for the helical tomotherapy (Tomotherapy). Planning of static gantry IMRT was performed with KonRad, Pinnacle and Panther DAO based on step&shoot IMRT delivery and Eclipse for sliding window IMRT. The prescribed doses for the high dose PTVs were 65.1Gy or 60.9Gy and for the low dose PTVs 55.8Gy or 52.5Gy dependend on resection status. Plan evaluation was based on target coverage, conformity and homogeneity, DVHs of OARs and the volume of normal tissue receiving more than 5Gy (V_5Gy). Additionally, the cumulative monitor units (MUs) and treatment times of the different technologies were compared. All evaluation parameters were averaged over all 10 patients for each technique and planning modality.</p> <p>Results</p> <p>Depending on IMRT technique and TPS, the mean CI values of all patients ranged from 1.17 to 2.82; and mean HI values varied from 0.05 to 0.10. The mean values of the median doses of the spared parotid were 26.5Gy for RapidArc and 23Gy for VMAT, 14.1Gy for Tomo. For fixed gantry techniques 21Gy was achieved for step&shoot+KonRad, 17.0Gy for step&shoot+Panther DAO, 23.3Gy for step&shoot+Pinnacle and 18.6Gy for sliding window.</p> <p>V_5Gyvalues were lowest for the sliding window IMRT technique (3499 ccm) and largest for RapidArc (5480 ccm). The lowest mean MU value of 408 was achieved by Panther DAO, compared to 1140 for sliding window IMRT.</p> <p>Conclusions</p> <p>All IMRT delivery technologies with their associated TPS provide plans with satisfying target coverage while at the same time respecting the defined OAR criteria. Sliding window IMRT, RapidArc and Tomo techniques resulted in better target dose homogeneity compared to VMAT and step&shoot IMRT. Rotational IMRT based on C-arm linacs and Tomotherapy seem to be advantageous with respect to OAR sparing and treatment delivery efficiency, at the cost of higher dose delivered to normal tissues. The overall treatment plan quality using Tomo seems to be better than the other TPS technology combinations.</p

Application of volumetric modulated arc therapy (VMAT) in a dual-vendor environment

Background and Purpose The purpose of this study was to assess plan quality and treatment time achievable with the new VMAT optimization tool implemented in the treatment planning system Oncentra MasterPlan® as compared to IMRT for Elekta SynergyS® linear accelerators. Materials and methods VMAT was implemented on a SynergyS® linear accelerator (Elekta Ltd., Crawley, UK) with Mosaiq® record and verify system (IMPAC Medical Systems, Sunnyvale, CA) and the treatment planning system Oncentra MasterPlan® (Nucletron BV, Veenendaal, the Netherlands). VMAT planning was conducted for three typical target types of prostate cancer, hypopharynx/larynx cancer and vertebral metastases, and compared to standard IMRT with respect to plan quality, number of monitor units (MU), and treatment time. Results For prostate cancer and vertebral metastases single arc VMAT led to similar plan quality as compared to IMRT. For treatment of the hypopharynx/larynx cancer, a second arc was necessary to achieve sufficient plan quality. Treatment time was reduced in all cases to 35% to 43% as compared to IMRT. Times required for optimization and dose calculation, however, increased by a factor of 5.0 to 6.8. Conclusion Similar or improved plan quality can be achieved with VMAT as compared to IMRT at reduced treatment times but increased calculation times